Research & development

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Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo
Scandi J of Immunology 69, 242–250 (2009)

Johnson E, Førland DT, Sætre L , Bernardshaw SV, Lyberg T and Hetland G

An immunostimulatory extract based on the medicinal mushroom Agaricus blazei Murill (AbM) has been shown to stimulate mononuclear phagocytes in vitro to produce pro-inflammatory cytokines, and to protect against lethal peritonitis in mice. The present aim was to study the effect of AbM on release of several cytokines in human whole blood both after stimulation ex vivo and in vivo after oral intake over several days in healthy volunteers. The 17 signal substances examined were; T helper 1 (Th1) cytokines [interleukin (IL)-2, interferon (IFN)-gamma and IL-12], T helper 2 cytokines (IL-4, IL-5 and IL-13), pleiotropic (IL-7, IL-17), pro-inflammatory [IL-1beta, IL-6, tumour necrosis factor (TNF)-alpha (mainly produced by Th1 cells)]–and anti-inflammatory (IL-10) cytokines, chemokines [IL-8, macrophage inhibitory protein (MIP)-1beta and monocyte chemoattractant protein (MCP)-1] and leukocyte growth factors [granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor]. After stimulation of whole blood ex vivo with 0.5-5.0% of a mushroom extract, AndoSan™ mainly containing AbM, there was a dose-dependent increase in all the cytokines studied, ranging from two to 399-fold (TNF-alpha). However, in vivo in the eight volunteers who completed the daily intake (60 ml) of this AbM extract for 12 days, a significant reduction was observed in levels of IL-1beta (97%), TNF-alpha (84%), IL-17 (50%) and IL-2 (46%). Although not significant, there was a trend towards reduced levels for IL-8, IFN-gamma and G-CSF, whilst those of the remaining nine cytokines tested, were unaltered. The discrepant results on cytokine release ex vivo and in vivo may partly be explained by the antioxidant activity of AbM in vivo and limited absorption of its large, complex and bioactive beta-glucans across the intestinal mucosa to the reticuloendothelial system and blood.

Effect of an Extract Based on the Medicinal Mushroom Agaricus blazei Murill on Expression of Cytokines and Calprotectin in Patients with Ulcerative Colitis and Crohn’s disease
Scand J of Immunology 73, 66–75 (2010)

Førland DT, Johnson E, Sætre L , Lyberg T , Lygren I and Hetland G

An immunomodulatory extract (AndoSan™) based on the medicinal mush-room Agaricus blazei Murill (AbM) has shown to reduce blood cytokine levels in healthy volunteers after 12 days’ ingestion, pointing to an anti-inflamma-tory effect. The aim was to study whether AndoSan™ had similar effects on cytokines in patients with ulcerative colitis (UC) and Crohn’s disease (CD). Calprotectin, a marker for inflammatory bowel disease (IBD), was also mea-sured. Patients with CD (n = 11) and with UC (n = 10) consumed 60 ml ⁄ day of AndoSan™. Patient blood plasma was harvested before and after 6 h LPS (1 ng ⁄ ml) stimulation ex vivo. Plasma and faecal calprotectin levels were analy-sed using ELISA and 17 cytokines [IL-2, IFN-c, IL-12 (Th1), IL-4, IL-5, IL-13 (Th2), IL-7, IL-17, IL-1b, IL-6, TNF-a, IL-8, MIP-1b, MCP-1, G-CSF, GM-CSF and IL-10] by multiplex assay. After 12 days’ ingestion of Ando-San, baseline plasma cytokine levels in UC was reduced for MCP-1 (40%) and in LPS-stimulated blood for MIP-1b (78%), IL-6 (44%), IL-1b (41%), IL-8 (30%), G-CSF (29%), MCP-1 (18%) and GM-CSF (17%). There were corresponding reductions in CD: IL-2 (100%), IL-17 (55%) and IL-8 (29%) and for IL-1b (35%), MIP-1b (30%), MCP-1 (22%), IL-8 (18%), IL-17 (17%) and G-CSF (14%), respectively. Baseline concentrations for the 17 cytokines in the UC and CD patient groups were largely similar. Faecal calprotectin was reduced in the UC group. Ingestion of an AbM-based medicinal mushroom by patients with IBD resulted in interesting anti-inflammatory effects as demon-strated by declined levels of pathogenic cytokines in blood and calprotectin in faeces.

Effect of a Medicinal Agaricus blazei Murill-Based Mushroom Extract, AndoSan™,on Symptoms, Fatigue and Quality of Life in Patients with Ulcerative Colitis in a Randomized Single-Blinded Placebo Controlled Study
PLoS ONE 11(3): e0150191. doi:10.1371/journal. pone.0150191 (2016)

Therkelsen SP, Hetland G, Lyberg T, Lygren I, Johnson E

50 patients with symptomatic ulcerative colitis were block-randomized and blinded for oral daily intake of AndoSan™ or placebo for the 21 days’ experimental period. The patients reported scores for symptoms, fatigue and health related quality of life (HRQoL) at days 0, 14 and 21. Fecal calprotectin and general blood parameters were also analyzed. In the AndoSan™ group (n = 24) symptoms improved from baseline (day 0) to days 14 and 21, with respective mean scores (95% CI) of 5.88 (4.92–6.83), 4.71 (3.90–5.52) (p = 0.002) and 4.50 (3.70–5.30) (p = 0.001). Corresponding improved mean scores (±SD) for total fatigue were 16.6 (5.59), 14.1 (4.50) (p = 0.001) and 15.1 (4.09) (p = 0.023). These scores in the placebo group (n = 26) were not improved. When comparing the two study groups using mixed model statistics, we found significant better scores for the AndoSan™-patients. HRQoL for dimensions bodily pain, vitality, social functioning and mental health improved in the AndoSan™ group. There were no alterations in general blood samples and fecal calprotectin.

An extract of the medicinal mushroom Agaricus blazei Murill can protect against allergy
Clin Mol Allergy 2009;7:6

Ellertsen LK, Hetland G

Agaricus blazei Murill (AbM) is an edible Brazilian mushroom that has been used in traditional medicine for a range of diseases. It has been shown to have anti-infection and anti-tumor properties in the mouse, which are due to induction of Th1 responses. On the other hand, IgE-mediated allergy is induced by a Th2 response. OBJECTIVE: Since according to the Th1/Th2 paradigm an increased Th1 response may promote a reduced Th2 response, the aim was to examine whether AbM had anti-allergy effects. METHODS: A mouse model for allergy was employed, in which the mice were immunized s.c. with the model allergen ovalbumin (OVA). Additionally, the animals were given a mushroom extract, AndoSan™, mainly (82%) containing AbM, but also Hericium erinaceum (15%) and Grifola frondosa (3%), or PBS p.o. either a day before or 19 days after the immunization. The mice were sacrificed on day 26, and anti-OVA IgE (Th2 response) and IgG2a (Th1 response) antibodies were examined in serum and Th1, Th2 and Treg cytokines in spleen cells cultures. RESULTS: It was found that the AndoSan™ extract both when given either before or after OVA immunization reduced the levels of anti-OVA IgE, but not IgG2a, in the mice. There was a tendency to reduced Th2 relative to Th1 cytokine levels in the AndoSan™ groups. CONCLUSION: This particular AbM extract may both prevent allergy development and be used as a therapeutical substance against established allergy.

An extract of the mushroom Agaricus blazei Murill administered orally protects against systemic Streptococcus pneumoniae infection in mice
Scand J Immunol 2005 Oct;62(4):393-8

Bernardshaw S, Johnson E, Hetland G

The aim was to investigate the antibacterial effect of the biologically active and edible mushroom Agaricus blazei Murill (AbM). A water extract of AbM or PBS control was administered orally before or with challenge to NIH/OlaHsd mice, experimentally infected intraperitoneally with the moderately virulent Streptococcus pneumoniae serotype 6B. End points were bacteraemia and survival rate. The AbM extract, protected against systemic S. pneumoniae 6B infection in the mice. It was most effective when given 24 h before inoculation but did also have protective effects when given together with challenge compared with control. The lack of antibiotic effect on pneumococci in vitro and increased levels of cytokines MIP-2 and TNF-alpha in the serum of mice receiving AbM extract, indicated that the protective effect of AbM was due to the involvement of the native immune system. This is the first report of anti-infection effects of AbM in vivo. Our results suggest that AbM extract may be useful as additional prophylactic and possibly therapeutic treatment against bacterial and possibly other infections in humans.

The Polar High Molecular Weight Fraction of the Agaricus blazei Murill Extract, AndoSan™, Reduces the Activity of the Tumor-Associated Protease, Legumain, in RAW 264.7 Cells
J Med Food 00 (0) 2014, 1–10

Berven L, Karppinen P, Hetland G and Samuelsen ABC

AndoSan™ is an extract of Agaricus blazei Murill (AbM; 82.4%), Hericium erinaceum (14.7%), and Grifola frondosa (2.9%). The main ingredient of AndoSan™, AbM, is rich in different forms of b-glucans. Since these exhibit potent antitumor activity and have immunomodulatory effects, the stimulatory effect of AndoSan™ on the production of different cytokines, chemokines, and leukocyte growth factors has predominantly been attributed to b-glucans. AndoSan™ has been claimed to consist of 90%carbohydrate, of which 2.8% is b-glucans, but in this study, we show that the carbohydrate content is only 2% of the dry weight, corresponding to 0.09% b-glucan per mL of AndoSan™. Fractionation of AndoSan™, followed by carbohydrate analysis and HPLC analysis revealed that most of the glucose was concentrated in the polar high molecular weight fraction of AndoSan™ (ethanol insoluble water extract [EIWE]-A) and that this extract was able to significantly inhibit the activity of the tumor-associated protease, legumain, in RAW 264.7 cells. Legumain is synthesized as a zymogen and undergoes pH-dependent autoactivation of the proform to reach an enzymatically active form. In this study, we demonstrate that both the polar and nonpolar AndoSan™ fractions are able to inhibit the autoactivation of prolegumain, and that the polar fractions of AndoSan™ are the most potent inhibitors of the active form of the enzyme.

Immunomodulatory Effects of the Agaricus blazei Murrill-Based Mushroom Extract AndoSan™ in Patients with Multiple Myeloma Undergoing High Dose Chemotherapy and Autologous Stem Cell Transplantation: A Randomized, Double Blinded Clinical Study
BioMed Research International 2015

Tangen JM, Tierens A, Caers J, Binsfeld M, Olstad OK, Trøseid AMS, Wang J, Geir Erland Tjønnfjord GE and Hetland G

Forty patients with multiple myeloma scheduled to undergo high dose chemotherapy with autologous stem cell support were randomized in a double blinded fashion to receive adjuvant treatment with the mushroom extract AndoSan™, containing 82% of Agaricus blazei Murrill (19 patients) or placebo (21 patients). Intake of the study product started on the day of stem cell mobilizing chemotherapy and continued until the end of aplasia after high dose chemotherapy, a period of about seven weeks. Thirty-three patients were evaluable for all study endpoints, while all 40 included patients were evaluable for survival endpoints. In the leukapheresis product harvested after stem cell mobilisation, increased percentages of Treg cells and plasmacytoid dendritic cells were found in patients receiving AndoSan™. Also, in this group, a significant increase of serum levels of IL-Ira, IL-5, and IL- 7 at the end of treatment was found. Whole genome microarray showed increased expression of immunoglobulin genes, Killer Immunoglobulin Receptor (KIR) genes, and HLA genes in the Agaricus group. Furthermore, AndoSan™ displayed a concentration dependent antiproliferative effect on mouse myeloma cells in vitro and displayed a concentration dependent antiproliferative effect on mouse myeloma.

The Agaricus blazei-Based Mushroom Extract, Andosan™, Protects against Intestinal Tumorigenesis in the A/J Min/+ Mouse
PLOS ONE DOI:10.1371/journal.pone.0167754 December 21, 2016

Hetland G, Eide DM, Tangen JM, Haugen MH, Mirlashari MR, Paulsen JE

The novel A/J Min/+ mouse, which is a model for human Familial Adenomatous Polyposis
(FAP), develops spontaneously multiple adenocarcinomas in the colon as well as in the small intestine. Agaricus blazei Murill (AbM) is an edible Basidiomycetes mushroom that has been used in traditional medicine against cancer and other diseases. The mushroom contains immunomodulating β-glucans and is shown to have antitumor effects in murine cancer models. Andosan™ is a water extract based on AbM (82%), but it also contains the medicinal Basidiomycetes mushrooms Hericeum erinaceus and Grifola frondosa.

Methods and findings
Tap water with 10% Andosan™ was provided as the only drinking water for 15 or 22 weeks to A/J Min/+ mice and A/J wild-type mice (one single-nucleotide polymorphism (SNP) difference), which then were exsanguinated and their intestines preserved in formaldehyde and the serum frozen. The intestines were examined blindly by microscopy and also stained for the tumor associated protease, legumain. Serum cytokines (pro- and anti-inflammatory, Th1-, Th2 -and Th17 type) were measured by Luminex multiplex analysis. Andosan™ treated A/J Min/+ mice had a significantly lower number of adenocarcinomas in the intestines, as well as a 60% significantly reduced intestinal tumor load (number of tumors x size) compared to control. There was also reduced legumain expression in intestines from Andosan™ treated animals. Moreover, Andosan™ had a significant cytotoxic effect correlating with apoptosis on the human cancer colon cell line, Caco-2, in vitro. When examining serum from both A/J Min/+ and wild type mice, there was a significant increase in anti-tumor Th1 type and pro-inflammatory cytokines in the Andosan™ treated mice.

The results from this mouse model for colorectal cancer shows significant protection of orally administered Andosan™ against development of intestinal cancer. This is supported by the finding of less legumain in intestines of Andosan™ treated mice and increased systemic Th1 cytokine response. The mechanism is probably both immuno-modulatory and growth inhibition of tumor cells by induction of apoptosis.

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